title: Regulation of L-type calcium channel sparklet activity by c-Src and PKC-α.
abstract: The activity of persistent Ca²⁺ sparklets, which are characterized by longer and more frequent channel open events than low-activity sparklets, contributes substantially to steady-state Ca²⁺ entry under physiological conditions. Here, we addressed two questions related to the regulation of Ca²⁺ sparklets by PKC-α and c-Src, both of which increase whole cell Cav1.2 current: 1) Does c-Src activation enhance persistent Ca²⁺ sparklet activity? 2) Does PKC-α activate c-Src to produce persistent Ca²⁺ sparklets? With the use of total internal reflection fluorescence microscopy, Ca²⁺ sparklets were recorded from voltage-clamped tsA-201 cells coexpressing wild-type (WT) or mutant Cav1.2c (the neuronal isoform of Cav1.2) constructs ± active or inactive PKC-α/c-Src. Cells expressing Cav1.2c exhibited both low-activity and persistent Ca²⁺ sparklets. Persistent Ca²⁺ sparklet activity was significantly reduced by acute application of the c-Src inhibitor PP2 or coexpression of kinase-dead c-Src. Cav1.2c constructs mutated at one of two COOH-terminal residues (Y²¹²²F and Y²¹³⁹F) were used to test the effect of blocking putative phosphorylation sites for c-Src. Expression of Y²¹²²F but not Y²¹³⁹F Cav1.2c abrogated the potentiating effect of c-Src on Ca²⁺ sparklet activity. We could not detect a significant change in persistent Ca²⁺ sparklet activity or density in cells coexpressing Cav1.2c + PKC-α, regardless of whether WT or Y²¹²²F Cav1.2c was used, or after PP2 application, suggesting that PKC-α does not act upstream of c-Src to produce persistent Ca²⁺ sparklets. However, our results indicate that persistent Ca²⁺ sparklet activity is promoted by the action of c-Src on residue Y²¹²² of the Cav1.2c COOH terminus.
authors.affiliation: Department of Biological Engineering University of Missouri, Columbia, Missouri, USA.
authors.forename: Jyoti
authors.initials: J
authors.lastname: Gulia
authors.forename: Manuel F
authors.initials: MF
authors.lastname: Navedo
authors.forename: Peichun
authors.initials: P
authors.lastname: Gui
authors.forename: Jun-Tzu
authors.initials: JT
authors.lastname: Chao
authors.forename: Jose L
authors.initials: JL
authors.lastname: Mercado
authors.forename: Luis F
authors.initials: LF
authors.lastname: Santana
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Davis
mesh_terms: D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism*; D002461:Cell Line, Transformed; D005786:Gene Expression Regulation; D006801:Humans; D008856:Microscopy, Fluorescence; D009154:Mutation; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D051571:Protein Kinase C-alpha / Q000235:genetics / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011743:Pyrimidines / Q000494:pharmacology; D051381:Rats; D015398:Signal Transduction; D014162:Transfection; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: C412373:AG 1879; D020746:Calcium Channels, L-Type; C419385:L-type calcium channel alpha(1C); D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; D051571:Protein Kinase C-alpha; D002118:Calcium
keywords: L-type calcium channel; PKC; TIRF microscopy; calcium entry; patch clamp